Regulated protein secretion is important for the establishment of cell polarity and is also important to present proteins on the cell surface during an immune response against tumorigenic viruses. To fully understand the process of regulated secretion, we need to understand the mechanisms used to transport proteins through the secretory pathway. No detailed mechanism of protein transport through late stages of the secretory pathway (i.e. Golgi to plasma membrane) is known. Long-term goals of this project are to determine all the components required for packaging, budding, sorting, and transport of Golgi-derived vesicles. The specific aims of this grant focus on the regulated transport of Fus1p, a Saccharomyces cerevisiae protein that localizes to the cell surface during mating. To identify proteins that function to transport Fus1p, we will use a candidate gene approach to identify mutations that cause Fus1p mislocalization. We will determine the plasma membrane localization signal on Fus1p through deletion and mutagenic PCR analysis. Finally, we will use a cell-free Golgi budding assay to determine if Fus1p is packaged into Golgi-derived vesicles and what the requirements are for Fus1p vesicle budding.